The EIF4EBP3 translational repressor is a marker of CDC73 tumor suppressor haploinsufficiency in a parathyroid cancer syndrome.

Germline mutation of the tumor suppressor gene CDC73 confers susceptibility to the hyperparathyroidism-jaw tumor syndrome associated with a high risk of parathyroid malignancy. Inactivating CDC73 mutations have also been implicated in sporadic parathyroid cancer, but are rare in sporadic benign parathyroid tumors. The molecular pathways that distinguish malignant from benign parathyroid transformation remain elusive. We previously showed that a hypomorphic allele of hyrax (hyx), the Drosophila homolog of CDC73, rescues the loss-of-ventral-eye phenotype of lobe, encoding the fly homolog of Akt1s1/ PRAS40. We report now an interaction between hyx and Tor, a central regulator of cell growth and autophagy, and show that eukaryotic translation initiation factor 4E-binding protein (EIF4EBP), a translational repressor and effector of mammalian target of rapamycin (mTOR), is a conserved target of hyx/CDC73. Flies heterozygous for Tor and hyx, but not Mnn1, the homolog of the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor associated with benign parathyroid tumors, are starvation resistant with reduced basal levels of Thor/4E-BP. Human peripheral blood cell levels of EIF4EBP3 were reduced in patients with CDC73, but not MEN1, heterozygosity. Chromatin immunoprecipitation demonstrated occupancy of EIF4EBP3 by endogenous parafibromin. These results show that EIF4EBP3 is a peripheral marker of CDC73 function distinct from MEN1-regulated pathways, and suggest a model whereby starvation resistance and/or translational de-repression contributes to parathyroid malignant transformation.

Cytoplasmic polyadenylation element binding protein is a conserved target of tumor suppressor HRPT2/CDC73.

Parafibromin, a tumor suppressor protein encoded by HRPT2/CDC73 and implicated in parathyroid cancer and the hyperparathyroidism-jaw tumor (HPT-JT) familial cancer syndrome, is part of the PAF1 transcriptional regulatory complex. Parafibromin has been implicated in apoptosis and growth arrest, but the mechanism by which its loss of function promotes neoplasia is poorly understood. In this study we report that a hypomorphic allele of hyrax (hyx), the Drosophila homolog of HRPT2/CDC73, rescues the loss-of-ventral-eye phenotype of lobe (Akt1s1). Such rescue is consistent with previous reports that hyx/parafibromin is required for the nuclear transduction of Wingless (Wg)/Wnt signals and that Wg signaling antagonizes lobe function. A screen using double hyx/lobe heterozygotes identified an additional interaction with orb and orb2, the homologs of mammalian cytoplasmic polyadenylation element binding protein (CPEB), a translational regulatory protein. Hyx and orb2 heterozygotes lived longer and were more resistant to starvation than controls. In mammalian cells, knockdown of parafibromin expression reduced levels of CPEB1. Chromatin immunoprecipitation (ChIP) showed occupancy of CPEB1 by endogenous parafibromin. Bioinformatic analysis revealed a significant overlap between human transcripts potentially regulated by parafibromin and CPEB. These results show that parafibromin may exert both transcriptional and, through CPEB, translational control over a subset of target genes and that loss of parafibromin (and CPEB) function may promote tumorigenesis in part by conferring resistance to nutritional stress.

HRPT2, encoding parafibromin, is mutated in hyperparathyroidism-jaw tumor syndrome.

We report here the identification of a gene associated with the hyperparathyroidism-jaw tumor (HPT-JT) syndrome. A single locus associated with HPT-JT (HRPT2) was previously mapped to chromosomal region 1q25-q32. We refined this region to a critical interval of 12 cM by genotyping in 26 affected kindreds. Using a positional candidate approach, we identified thirteen different heterozygous, germline, inactivating mutations in a single gene in fourteen families with HPT-JT. The proposed role of HRPT2 as a tumor suppressor was supported by mutation screening in 48 parathyroid adenomas with cystic features, which identified three somatic inactivating mutations, all located in exon 1. None of these mutations were detected in normal controls, and all were predicted to cause deficient or impaired protein function. HRPT2 is a ubiquitously expressed, evolutionarily conserved gene encoding a predicted protein of 531 amino acids, for which we propose the name parafibromin. Our findings suggest that HRPT2 is a tumor-suppressor gene, the inactivation of which is directly involved in predisposition to HPT-JT and in development of some sporadic parathyroid tumors.

Reoperation for hyperparathyroidism in multiple endocrine neoplasia type 1.

BACKGROUND: Patients with multiple endocrine neoplasia type 1 and hyperparathyroidism often undergo multiple operations because of inadequate initial surgery, presence of supernumerary and ectopic glands, regrowth of remnant glands, or autograft hyperfunction. Management of this patient population is complex. METHODS: From January 1975 to December 2000 we performed 94 reoperative parathyroidectomies consisting of 79 neck reexplorations, 12 autograft removals, and 3 median sternotomies in 75 patients. Data were gathered by retrospective chart review and follow-up telephone interviews. RESULTS: Excluding autograft excision, reoperative surgery was successful (normocalcemia longer than 6 months) in 91%; autograft removal was successful in only 58%. With a median follow-up of 59 months, 64% of patients are currently free from hypercalcemia, and this outcome was not influenced by the total number of glands resected. The median time to recurrent hypercalcemia was 125 months. Thirty patients received an autograft after reoperation. The complication rate for all reoperations was 12%, including permanent recurrent laryngeal nerve injury in 2 patients (2.1%). CONCLUSIONS: Reoperative parathyroidectomy in patients with multiple endocrine neoplasia type 1 was safe and successful in the majority of patients; however, recurrent hyperparathyroidism is likely to develop in most individuals beyond 10 years of follow-up. The total number of glands accounted for after reoperation is not associated with successful outcome.

G alpha(q/11) coupled to mammalian phospholipase C beta 3-like enzyme mediates the ginsenoside effect on Ca(2+)-activated Cl(-) current in the Xenopus oocyte.

Recently we demonstrated that ginsenosides, the active ingredients of Panax ginseng, enhanced Ca(2+)-activated Cl(-) current in the Xenopus oocyte through a signal transduction mechanism involving the activation of pertussis toxin-insensitive G protein and phospholipase C (PLC). However, it has not yet been determined precisely which G protein subunit(s) and which PLC isoform(s) participate in the ginsenoside signaling. To provide answers to these questions, we investigated the changes in ginsenoside effect on the Cl(-) current after intraoocyte injections of the cRNAs coding various G protein subunits, a regulator of G protein signaling (RGS2), and G beta gamma-binding proteins. In addition, we examined which of mammalian PLC beta 1-3 antibodies injected into the oocyte inhibited the action of ginsenosides on the Cl(-) current. Injection of G alpha(q) or G alpha(11) cRNA increased the basal Cl(-) current recorded 48 h after, and it further prevented ginsenosides from enhancing the Cl(-) current, whereas G alpha(i2) and G alpha(oA) cRNA injection had no significant effect. The changes following G alpha(q) cRNA injection were prevented when G beta(1)gamma(2) and G alpha(q) subunits were co-expressed by simultaneous injection of the cRNAs coding these subunits. Injection of cRNA coding G alpha(q)Q209L, a constitutively active mutant that does not bind to G beta gamma, produced effects similar to those of G alpha(q) cRNA injection. The effects of G alpha(q)Q209L cRNA injection, however, were not prevented by co-injection of G beta(1)gamma(2) cRNA. Injection of the cRNA coding RGS2, which interacts most selectively with G alpha(q/11) among various identified RGS isoforms and stimulates the hydrolysis of GTP to GDP in active GTP-bound G alpha subunit, resulted in a severe attenuation of ginsenoside effect on the Cl(-) current. Finally, antibodies against PLC beta 3, but not -beta 1 and -beta 2, markedly attenuated the ginsenoside effect examined at 3-h postinjection. These results suggest that G alpha(q/11) coupled to mammalian PLC beta 3-like enzyme mediates ginsenoside effect on Ca(2+)-activated Cl(-) current in the Xenopus oocyte.

Gbetagamma affinity for bovine rhodopsin is determined by the carboxyl-terminal sequences of the gamma subunit.

Two native betagamma dimers, beta(1)gamma(1) and beta(1)gamma(2), display very different affinities for receptors. Since these gamma subunits differ in both primary structure and isoprenoid modification, we examined the relative contributions of each to Gbetagamma interaction with receptors. We constructed baculoviruses encoding gamma(1) and gamma(2) subunits with altered CAAX (where A is an aliphatic amino acid) motifs to direct alternate or no prenylation of the gamma chains and a set of gamma(1) and gamma(2) chimeras with the gamma(2) CAAX motif at the carboxyl terminus. All the gamma constructs coexpressed with beta(1) in Sf9 cells yielded beta(1)gamma dimers, which were purified to near homogeneity, and their affinities for receptors and Galpha were quantitatively determined. Whereas alteration of the isoprenoid of gamma(1) from farnesyl to geranylgeranyl and of gamma(2) from geranylgeranyl to farnesyl had no impact on the affinities of beta(1)gamma dimers for Galpha(t), the non-prenylated beta(1)gamma(2) dimer had significantly diminished affinity. Altered prenylation resulted in a <2-fold decrease in affinity of the beta(1)gamma(2) dimer for rhodopsin and a <3-fold change for the beta(1)gamma(1) dimer. In each case with identical isoprenylation, the beta(1)gamma(2) dimer displayed significantly greater affinity for rhodopsin compared with the beta(1)gamma(1) dimer. Furthermore, dimers containing chimeric Ggamma chains with identical geranylgeranyl modification displayed rhodopsin affinities largely determined by the carboxyl-terminal one-third of the protein. These results indicate that isoprenoid modification of the Ggamma subunit is essential for binding to both Galpha and receptors. The isoprenoid type influences the binding affinity for receptors, but not for Galpha. Finally, the primary structure of the Ggamma subunit provides a major contribution to receptor binding of Gbetagamma, with the carboxyl-terminal sequence conferring receptor selectivity.

RGS9-G beta 5 substrate selectivity in photoreceptors. Opposing effects of constituent domains yield high affinity of RGS interaction with the G protein-effector complex.

RGS proteins regulate the duration of G protein signaling by increasing the rate of GTP hydrolysis on G protein alpha subunits. The complex of RGS9 with type 5 G protein beta subunit (G beta 5) is abundant in photoreceptors, where it stimulates the GTPase activity of transducin. An important functional feature of RGS9-G beta 5 is its ability to activate transducin GTPase much more efficiently after transducin binds to its effector, cGMP phosphodiesterase. Here we show that different domains of RGS9-G beta 5 make opposite contributions toward this selectivity. G beta 5 bound to the G protein gamma subunit-like domain of RGS9 acts to reduce RGS9 affinity for transducin, whereas other structures restore this affinity specifically for the transducin-phosphodiesterase complex. We suggest that this mechanism may serve as a general principle conferring specificity of RGS protein action.

Multiple endocrine neoplasia type 1: new clinical and basic findings.

Multiple endocrine neoplasia type 1 (MEN1) provides a prime example of how a rare disease can advance our understanding of basic cell biology, neoplasia and common endocrine tumors. MEN1 is expressed mainly as parathyroid, enteropancreatic neuroendocrine, anterior pituitary and foregut carcinoid tumors. It is an autosomal dominant disease caused by mutation of the MEN1 gene. Since its identification, the MEN1 gene has been implicated in many common endocrine and non-endocrine tumors. This is a brief overview of recent scientific advances relating to MEN1, including newly recognized clinical features that are now better characterized by genetic analysis, insights into the function of the MEN1-encoded protein menin, and refined recommendations for mutation testing and tumor screening, which highlight our increasing understanding of this complex syndrome.

Nuclear localization of G protein beta 5 and regulator of G protein signaling 7 in neurons and brain.

The role that Gbeta(5) regulator of G protein signaling (RGS) complexes play in signal transduction in brain remains unknown. The subcellular localization of Gbeta(5) and RGS7 was examined in rat PC12 pheochromocytoma cells and mouse brain. Both nuclear and cytosolic localization of Gbeta(5) and RGS7 was evident in PC12 cells by immunocytochemical staining. Subcellular fractionation of PC12 cells demonstrated Gbeta(5) immunoreactivity in the membrane, cytosolic, and nuclear fractions. Analysis by limited proteolysis confirmed the identity of Gbeta(5) in the nuclear fraction. Subcellular fractionation of mouse brain demonstrated Gbeta(5) and RGS7 but not Ggamma(2/3) immunoreactivity in the nuclear fraction. RGS7 and Gbeta(5) were tightly complexed in the brain nuclear extract as evidenced by their coimmunoprecipitation with anti-RGS7 antibodies. Chimeric protein constructs containing green fluorescent protein fused to wild-type Gbeta(5) but not green fluorescent fusion proteins with Gbeta(1) or a mutant Gbeta(5) impaired in its ability to bind to RGS7 demonstrated nuclear localization in transfected PC12 cells. These findings suggest that Gbeta(5) undergoes nuclear translocation in neurons via an RGS-dependent mechanism. The novel intracellular distribution of Gbeta(5).RGS protein complexes suggests a potential role in neurons communicating between classical heterotrimeric G protein subunits and/or their effectors at the plasma membrane and the cell nucleus.

Ultrasound and sestamibi scan as the only preoperative imaging tests in reoperation for parathyroid adenomas.

BACKGROUND: In an effort to determine an efficient algorithm for the evaluation of patients with parathyroid adenomas in the reoperative setting, we explored the combination of using ultrasound scans (US) and sestamibi scintigraphy as the only preoperative imaging tests. METHODS: We analyzed the outcomes of 62 consecutive patients who were treated between January 1995 and May 1999 and who were referred for persistent primary hyperparathyroidism after initial surgical exploration, at which time no abnormal parathyroid glands had been found. Although all patients underwent US, computed tomography scan, magnetic resonance imaging, and sestamibi scan, we analyzed the success of localization and reoperation using only the results of US and sestamibi scan. RESULTS: Sixty-one patients (98%) underwent curative reoperations. The sensitivity, positive predictive value, and accuracy for US were 90%, 86%, and 84%, respectively; the corresponding values for sestamibi imaging were 78%, 94%, and 74%, respectively. In 58 of 62 cases (94%) preoperative US and/or sestamibi scan accurately identified the adenoma. In 3 patients for whom combined US and sestamibi scan were inaccurate, 1 adenoma was found by intraoperative US in the strap muscle; 1 adenoma was found by blind cervical thymectomy, and 1 adenoma was found by planned sternotomy that was based on computed tomography findings. CONCLUSIONS: This study supports an algorithm of obtaining US and sestamibi scan as the initial and perhaps only preoperative localization tests for patients with primary hyperparathyroidism after failed operation, at which time no abnormal glands had been found.

Dual regulation of Akt/protein kinase B by heterotrimeric G protein subunits.

While positive regulation of c-Akt (also known as protein kinase B) by receptor tyrosine kinases is well documented, compounds acting through G protein-coupled receptors can also activate Akt and its downstream targets. We therefore explored the role of G protein subunits in the regulation of Akt in cultured mammalian cells. In HEK-293 and COS-7 cells transiently transfected with beta(2)-adrenergic or m2 muscarinic receptors, respectively, treatment with agonist-induced phosphorylation of Akt at serine 473 as evidenced by phosphoserine-specific immunoblots. This effect was blocked by the phosphatidylinositol-3-OH kinase inhibitor LY294002 and wild-type Galpha(i1), and was not duplicated by co-transfection of the constitutively active Galpha(s)-Q227L or Galpha(i)-Q204L mutant. Co-transfection of Gbeta(1), Gbeta(2) but not Gbeta(5) together with Ggamma(2) activated the kinase when assayed in vitro following immunoprecipitation of the epitope-tagged enzyme. In contrast, constitutively activated G protein subunits representing the four Galpha subfamilies were found unable to activate Akt in either cell line. The latter results are in disagreement with a report by Murga et al. (Murga, C., Laguinge, L., Wetzker, R., Cuadrado, A., and Gutkind, J. S. (1998) J. Biol. Chem. 273, 19080-19085) that described activation of Akt in response to mutationally activated Galpha(q) and Galpha(i) transfection in COS cells. To the contrary, in our experiments Galpha(q)-Q209L inhibited Akt activation resulting from betagamma or mutationally activated H-Ras co-transfection in these cells. In HEK-293 cells Galpha(q)-Q209L transfection inhibited insulin-like growth factor-1 activation of epitope-tagged Akt. In m1 muscarinic receptor transfected HEK-293 cells, carbachol inhibited insulin-like growth factor-1 stimulated phosphorylation at Ser(473) of endogenous Akt in an atropine-reversible fashion. We conclude that G proteins can regulate Akt by two distinct and potentially opposing mechanisms: activation by Gbetagamma heterodimers in a phosphatidylinositol-3-OH kinase-dependent fashion, and inhibition mediated by Galpha(q). This work identifies Akt as a novel point of convergence between disparate signaling pathways.

Differential expression of the G protein beta(5) gene: analysis of mouse brain, peripheral tissues, and cultured cell lines.

A neurally expressed heterotrimeric G protein beta subunit, Gbeta(5), has been found to exhibit functional specialization with respect to its interactions with effector targets and Galpha subunits. A splice variant of Gbeta(5) that contains an N-terminal 42-residue extension, Gbeta(5)-long, has been described in the retina. To define better the potential range of its specialized interactions, analysis of Gbeta(5) gene transcript and protein expression in mouse brain and other tissues and cell lines was performed. Quantification by ribonuclease protection assay of Gbeta(5) transcript expression in the developing brain demonstrates a fivefold increase that occurs postnatally. Analysis of transcript expression by in situ hybridization and ribonuclease protection assay indicates that the Gbeta(5) gene is differentially expressed among multiple adult mouse brain regions, including the motor and occipital cortex, the olfactory bulb and associated rhinencephalic structures, hypothalamus, pontine cochlear nuclei, and Purkinje cells in the cerebellum. Gbeta(5) is also expressed in several cultured cell lines of neuroendocrine origin, including murine alphaT3-1 pituitary gonadotrophs and GT1-7 hypothalamic cells, and rat PC12 pheochromocytoma cells. Immunoblotting of tissue homogenates with antibodies to two peptides common to Gbeta(5) and Gbeta(5)-long confirmed expression of Gbeta(5) in multiple brain regions and in spinal cord and expression of Gbeta(5)-long in retina. Taken together, these results suggest that the specialized molecular properties of Gbeta(5) have been adapted to diverse neural functions in the adult brain.

New dimensions in G protein signalling: G beta 5 and the RGS proteins.

The beta gamma complex of G-proteins regulates effectors independently of the G alpha subunits, such that upon activation G proteins give may signal downstream along one or both pathways. The G beta 5 isoform exhibits much less homology with other G beta isoforms (approximately 50%) and is preferentially expressed in brain. The G beta 5 isoform exhibits novel properties in its activation of effector pathways such as MAPK, phospholipase C-beta, and adenylyl cyclase type II when compared to G beta 1. Recently specific native complexes between G beta 5 and the regulator of G protein signaling (RGS) protein-7 (RGS7) and between G beta 5L (a splice variant with a 42 amino acid N-terminal extension) and RGS9 have been isolated from different retinal fractions. Such findings are not accounted for by current models as only the G alpha subunits and not G beta had been previously implicated in RGS protein function. These recent novel observations further reinforce the view of G beta 5 as a unique and highly specialized G protein subunit.

Regulation of NHE3 activity by G protein subunits in renal brush-border membranes.

NHE3 activity is regulated by phosphorylation/dephosphorylation processes and membrane recycling in intact cells. However, the Na(+)/H(+) exchanger (NHE) can also be regulated by G proteins independent of cytoplasmic second messengers, but the G protein subunits involved in this regulation are not known. Therefore, we studied G protein subunit regulation of NHE3 activity in renal brush-border membrane vesicles (BBMV) in a system devoid of cytoplasmic components and second messengers. Basal NHE3 activity was not regulated by G(s)alpha or G(i)alpha, because antibodies to these G proteins by themselves were without effect. The inhibitory effect of D(1)-like agonists on NHE3 activity was mediated, in part, by G(s)alpha, because it was partially reversed by anti-G(s)alpha antibodies. Moreover, the amount of G(s)alpha that coimmunoprecipitated with NHE3 was increased by fenoldopam in both brush-border membranes and renal proximal tubule cells. Furthermore, guanosine 5'-O-(3-thiotriphosphate) but not guanosine 5'-O-(2-thiodiphosphate), the inactive analog of GDP, increased the amount of G(s)alpha that coimmunoprecipitated with NHE3. The alpha(2)-adrenergic agonist, UK-14304 or pertussis toxin (PTX) alone had no effect on NHE3 activity, but UK-14304 and PTX treatment attenuated the D(1)-like receptor-mediated NHE3 inhibition. The ability of UK-14304 to attenuate the D(1)-like agonist effect was not due to G(i)alpha, because the attenuation was not blocked by anti-G(i)alpha antibodies or by PTX. Anti-Gbeta(common) antibodies, by themselves, slightly inhibited NHE3 activity but had little effect on D(1)-like receptor-mediated NHE3 inhibition. However, anti-Gbeta(common) antibodies reversed the effects of UK-14304 and PTX on D(1)-like agonist-mediated NHE3 inhibition. These studies provide concrete evidence of a direct regulatory role for G(s)alpha, independent of second messengers, in the D(1)-like-mediated inhibition of NHE3 activity in rat renal BBMV. In addition, beta/gamma dimers of heterotrimeric G proteins appear to have a stimulatory effect on NHE3 activity in BBMV.

Alterations in detergent solubility of heterotrimeric G proteins after chronic activation of G(i/o)-coupled receptors: changes in detergent solubility are in correlation with onset of adenylyl cyclase superactivation.

Prolonged G(i/o) protein-coupled receptor activation has been shown to lead to receptor internalization and receptor desensitization. In addition, it is well established that although acute activation of these receptors leads to inhibition of adenylyl cyclase (AC), long-term activation results in increased AC activity (especially evident on removal of the inhibitory agonist), a phenomenon defined as AC superactivation or sensitization. Herein, we show that chronic exposure to agonists of G(i)-coupled receptors also leads to a decrease in cholate detergent solubility of G protein subunits, and that antagonist treatment after such chronic agonist exposure leads to a time-dependent reversal of the cholate insolubility. With Chinese hamster ovary and COS cells transfected with several G(i/o)-coupled receptors (i.e., mu- and kappa-opioid, and m(4)-muscarinic), we observed that although no overall change occurred in total content of G(alphai)- and beta(1)-subunits, chronic agonist treatment led to a marked reduction in the ability of 1% cholate to solubilize G(betagamma) as well as G(alphai). This solubility shift is exclusively observed with G(alphai), and was not seen with G(alphas). The disappearance and reappearance of G(alphai) and G(betagamma) subunits from and to the detergent-soluble fractions occur with similar time courses as observed for the onset and disappearance of AC superactivation. Lastly, pertussis toxin, which blocks acute and chronic agonist-induced AC inhibition and superactivation, also blocks the shift in detergent solubility. These results suggest a correlation between the solubility shift of the heterotrimeric G(i) protein and the generation of AC superactivation.

Copurification of brain G-protein beta5 with RGS6 and RGS7.

A structurally divergent G-protein beta subunit expressed in brain and retina, Gbeta5, exhibits functional specialization in its protein-protein interactions in vitro. In retina, Gbeta5 has been isolated in a soluble complex with regulator of G-protein signaling RGS7. The function and molecular associations of Gbeta5 in brain are unknown. To identify tightly bound proteins associated with Gbeta5 in the brain, it was immunoaffinity-purified from a nonionic detergent extract of washed mouse brain membranes using an antibody directed against its N terminus. Elution with cognate peptide revealed a broad band of 55 kDa that coeluted with Gbeta5 on SDS-PAGE. The copurifying 55 kDa band was identified as an approximately 1:1 mixture of RGS6 and RGS7 by matrix-assisted laser desorption ionization mass spectroscopic analysis of tryptic peptides. Gbeta5 and RGS7 could be reciprocally coimmunoprecipitated from unfractionated brain membrane extracts confirming the tight association of native proteins. In contrast, immunoblotting of the peptide eluate revealed no copurifying Galphaq/11, Galphai1/2, Ggamma2, Ggamma3, or Ggamma7. These findings implicate RGS6 and RGS7 in the function of Gbeta5 in the brain and suggest that a large fraction of membrane-targeted Gbeta5 has no associated G subunit and therefore functions outside the canonical framework of G(beta)(gamma) interactions.

G protein regulation of adenylate cyclase.

Adenylate cyclase integrates positive and negative signals that act through G protein-coupled cell-surface receptors with other extracellular stimuli to finely regulate levels of cAMP within the cell. Recently, the structures of the cyclase catalytic core complexed with the plant diterpene forskolin, and a cyclase-forskolin complex bound to an activated form of the stimulatory G protein subunit Gs alpha have been solved by X-ray crystallography. These structures provide a wealth of detail about how different signals could converge at the core cyclase domains to regulate catalysis. In this article, William Simonds reviews recent advances in the molecular and structural biology of this key regulatory enzyme, which provide new insight into its ability to integrate multiple signals in diverse cellular contexts.

Inhibition of adenylyl cyclase isoforms V and VI by various Gbetagamma subunits.

An intriguing development in the G-protein signaling field has been the finding that not only the Galpha subunit, but also Gbetagamma subunits, affect a number of downstream target molecules. One of the downstream targets of Gbetagamma is adenylyl cyclase, and it has been demonstrated that a number of isoforms of adenylyl cyclase can be either inhibited or stimulated by Gbetagamma subunits. Until now, adenylyl cyclase type I has been the only isoform reported to be inhibited by free Gbetagamma. Here we show by transient cotransfection into COS-7 cells of either adenylyl cyclase V or VI, together with Ggamma2 and various Gbeta subunits, that these two adenylyl cyclase isozymes are markedly inhibited by Gbetagamma. In addition, we show that Gbeta1 and Gbeta5 subunits differ in their activity. Gbeta1 transfected alone markedly inhibited adenylyl cylcase V and VI (probably by recruiting endogenous Ggamma subunits). On the other hand, Gbeta5 produced less inhibition of these isozymes, and its activity was enhanced by the addition of Ggamma2. These results demonstrate that adenylyl cyclase types V and VI are inhibited by Gbetagamma dimers and that Gbeta1 and Gbeta5 subunits differ in their capacity to regulate these adenylyl cyclase isozymes.

Differential modulation of adenylyl cyclases I and II by various G beta subunits.

The accepted dogma concerning the regulation of adenylyl cyclase (AC) activity by G beta gamma dimers states that the various isoforms of AC respond differently to the presence of free G beta gamma. It has been demonstrated that AC I activity is inhibited and AC II activity is stimulated by G beta gamma subunits. This result does not address the possible differences in modulation that may exist among the different G beta gamma heterodimers. Six isoforms of G beta and 12 isoforms of G gamma have been cloned to date. We have established a cell transfection system in which G beta and G gamma cDNAs were cotransfected with either AC isoform I or II and the activity of these isoforms was determined. We found that while AC I activity was inhibited by both G beta 1/gamma 2 and G beta 5/gamma 2 combinations, AC II responded differentially and was stimulated by G beta 1/gamma 2 and inhibited by G beta 5/gamma 2. This finding demonstrates differential modulatory activity by different combinations of G beta gamma on the same AC isoform and demonstrates another level of complexity within the AC signaling system.

The coiled-coil region of the G protein beta subunit. Mutational analysis of Ggamma and effector interactions.

The beta and gamma subunits of the heterotrimeric G proteins remain tightly associated throughout the signaling cycle as the betagamma dimer interacts with Galpha, receptors, and effectors. A coiled-coil structure involving alpha-helical segments at the N termini of the beta and gamma subunits contributes to the dimerization interface and has been implicated in effector signaling in yeast. Scanning mutagenesis of the coiled-coil region of the mammalian beta1 subunit was performed to examine the effect of point mutations on betagamma assembly and effector signaling in COS cell cotransfection assays. In addition to the E10K mutation described previously, mutations A11E, L14E, and I18E in beta1 were found to block betagamma association, as evidenced by the failure of the Gbeta mutants to undergo cytosolic translocation with cotransfected nonisoprenylated Ggamma. Although none of 14 beta1 point mutations prevented the betagamma-dependent activation of the c-Jun N-terminal kinase (JNK) effector pathway, the D20K point mutation enhanced JNK but not phospholipase C-beta2 activation. These findings implicate the coiled-coil region of Gbeta in JNK signaling, provide further evidence that the structural features of the betagamma complex mediating effector regulation may differ among effectors, and identify single codons in the mammalian beta subunit where mutation might yield a phenotype of defective signal transduction.